Abstract Rotavirus (RV) infection causes life-threatening, dehydrating diarrhea and is the leading cause of diarrheal deaths among children <5 years old despite availability of a vaccine. Critically, the oral vaccine is less effective in middle- and low-income countries where disproportionately more deaths occur compared to high-income countries. Addressing this disparity in vaccine effectiveness is a major public health priority. Correlates of protection do not exist, and cellular responses against RV in humans remain incompletely understood. Mounting evidence supports a direct role for the gut microbiota in modulating humoral and cellular immune responses to oral vaccines, but little is known about their actual mechanism of action. In our pilot study, vaccine responders had a significantly greater abundance of Bifidobacterium longum and higher content of microbial genes associated with folate transformation in their gut compared to nonresponders. These data suggest that infants may depend on microbes such as B. longum to synthesize folate de novo as a mechanism for RV-specific immune cell expansion. We hypothesize that de novo folate synthesis by microbes such as B. longum facilitates RV-specific immune cell expansion, and that levels of folate modulate vaccine immunogenicity. We propose to study 330 infants from the US, Panama, and Peru where vaccine efficacy is known to be high, medium and low, respectively, by using both stored and prospectively collected longitudinal samples of blood and stool from infants 0 to 12 months of age. We have designed a novel RV ?megapool? of immunogenic peptides to define cellular immune responses to RV vaccination in addition to assessing traditional serum RV-specific IgA and stool RV shedding after immunization (Aim 1). We will characterize gut microbial composition and function using metagenomic sequencing at multiple pre-vaccination time points in vaccine responders and nonresponders to determine if the abundance of B. longum and capacity to synthesize folate predict vaccine immunogenicity (Aim 2). We will analyze the metabolic byproducts to identify if folate or other metabolites enhance vaccine response (Aim 3). Our unique team of experts in vaccinology, immunology, microbiology, biochemistry, and bioinformatics will ensure successful integrative analysis and interpretation of these immunologic and multi-omics data. Completion of the study will provide a comprehensive characterization of microbial and metabolic biomarkers of RV vaccine responses, paving the way for targeted immune augmentation strategies.